Acetylenic acids inhibiting azole-resistant Candida albicans from Pentagonia gigantifolia

Article abstract of DOI:10.1021/np030196r

Antifungal bioassay-guided isolation of the ethanol extract of the roots of Pentagonia gigantifolia yielded 6-octadecynoic acid (1) and the new 6-nonadecynoic acid (2). Compounds 1 and 2 inhibited the growth of fluconazole-susceptible and -resistant Candida albicans strains. Their antifungal potencies were comparable to those of amphotericin B and fluconazole. Of particular significance is the low cytotoxicity and specific activity of 1 and 2 against C. albicans.

Full text of DOI:10.1021/np030196r

1
132
J . Nat. Prod. 2003, 66, 1132-1135
Acetylen ic Acid s In h ibitin g Azole-Resista n t Ca n d id a a lbica n s fr om
P en ta gon ia giga n tifolia
,
†
†
†
‡
†
†,§
Xing-Cong Li,* Melissa R. J acob, Hala N. ElSohly, Dale G. Nagle, Troy J . Smillie, Larry A. Walker, and
Alice M. Clark*,
†,‡,⊥
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, Department of Pharmacognosy
and Department of Pharmacology, School of Pharmacy, The University of Mississippi, University, Mississippi 38677
Received April 30, 2003
Antifungal bioassay-guided isolation of the ethanol extract of the roots of Pentagonia gigantifolia yielded
6
-octadecynoic acid (1) and the new 6-nonadecynoic acid (2). Compounds 1 and 2 inhibited the growth of
fluconazole-susceptible and -resistant Candida albicans strains. Their antifungal potencies were
comparable to those of amphotericin B and fluconazole. Of particular significance is the low cytotoxicity
and specific activity of 1 and 2 against C. albicans.
Candida albicans, a component of the normal human
microflora, is the most common cause of fungal infections,
and Candida species are the fourth leading cause of
The ethanol extract of the roots of P. gigantifolia was
first fractionated by silica gel column chromatography. The
most active fraction was further chromatographed on
reversed-phase silica gel C18 using aqueous methanol to
afford compounds 1 and 2 in yields of 0.39% and 0.088%,
respectively, of the dried plant material.
Compound 1 was identified as 6-octadecynoic acid (tariric
acid) by GC-MS analysis of its methyl ester (1a ) via a
direct library search. The structure of 1 was further
1
nosocomial infection in the United States. Studies have
further shown2 that C. albicans can become transiently
resistant to azole drugs rapidly after exposure to the
antifungal drug fluconazole, which involves inhibition of
ergosterol biosynthesis via interaction with the fungal
3
enzyme 14R-lanosterol demethylase. Mechanisms of azole
1
13
resistance that have been identified include alterations in
the gene encoding the target enzyme or overexpression of
efflux pump (CDR or MDR) genes.3 The development of
fluconazole resistance in C. albicans has been well docu-
mented for patients with AIDS and recurrent oropharyn-
geal candidiasis.2 Therefore, there is an urgent need to
discover new prototype antibiotics to combat these resistant
pathogens.
supported by its high-resolution ESIMS and H and
C
1
13
NMR data. Most H and C NMR signals of 1 were
assigned via 2D NMR techniques and are listed in the
Experimental Section since there are only low-resolution
,4
1
6
H NMR data (60 MHz) available for this compound.
The molecular formula of compound 2, C19 , was
established by a combination of high-resolution ESIMS and
,5
32 2
H O
13C NMR spectra. The H and 13C NMR spectra of 2 were
1
In our continued search for prototype antifungal agents
from higher plants, the ethanol extract of the roots of a
Peruvian plant, Pentagonia gigantifolia Ducke (Rubiaceae),
demonstrated significant antifungal activity against C. al-
bicans (IC50 < 20 µg/mL). However, no chemical or biologi-
cal studies have been reported on the plants of the genus
Pentagonia. A subsequent bioassay-guided fractionation
and purification of the active extract led to the identifica-
tion of two acetylenic acids, 6-octadecynoic acid (1) and the
new 6-nonadecynoic acid (2). The two compounds showed
potent antifungal activity against C. albicans and a series
of fluconazole-resistant C. albicans strains. In this paper,
we document the isolation, structure identification, and
antifungal activity of these two compounds.
in good agreement with those of 1, displaying characteristic
signals for a triple bond and a carboxylic group. The only
difference between their 13C NMR spectra is that 2 pos-
2
sessed an additional carbon signal around δ 30.9 (CH ),
1
while in the H NMR spectrum it showed two additional
protons in the region δ 1.25-1.37 when the integration
area was compared with that of 1. This indicated that
compound 2 is a homologue of 1. The methyl ester of 2 (2a )
showed a fragmentation pattern similar to 1a by GC-MS
analysis. The intense fragment ion at m/z 154 in both 1a
and 2a was diagnostic of the presence of a triple bond at
C-6 position.7 Further support was derived from a DQF-
COSY experiment, which clearly identified a coupling
network through H-2 (δ 2.38) f H-3 (δ 1.74) f H-4 (δ 1.54)
f H-5 (δ 2.18). H-5 was in turn coupled with H-8 (δ 2.13)
through a 1,4-long-range coupling (J ) 2.2 Hz), which is
characteristic of acetylenic compounds. Other 2D NMR
techniques, including HMQC and HMBC, were employed
to confirm the straight chain structure and facilitate the
assignment of its NMR signals. Therefore, the structure
of compound 2 was established as 6-nonadecynoic acid.
,8
When examined for antifungal activity against Candida
albicans, Cryptococcus neoformans, and Aspergillus fumi-
gatus and for antibacterial activity against Staphylococcus
aureus, methicillin-resistant S. aureus, Pseudomonas aerug-
inosa, and Mycobacterium intracellulare, compounds 1 and
*
To whom correspondence should be addressed. Tel: 662-915-6742.
Fax: 662-915-1006. E-mail (X.-C.L.): xli@olemiss.edu. E-mail (A.M.C.):
amc@olemiss.edu.
2
were active only against C. albicans. The antifungal
†
potencies of 1 and 2 against C. albicans were measured
National Center for Natural Products Research.
Department of Pharmacognosy.
Department of Pharmacology.
Current address: Office of Research, 125 Old Chemistry, The Univer-
‡
9
using a modified version of the NCCLS method and
§
⊥
comparable to the positive controls of three classes of
antifungal drugs, amphotericin B, fluconazole, and flucy-
sity of Mississippi, University, MS 38677.
1
0.1021/np030196r CCC: $25.00
© 2003 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/29/2003

Notes
J ournal of Natural Products, 2003, Vol. 66, No. 8 1133
Exp er im en ta l Section
Ta ble 1. Antifungal Activity of Compounds 1 and 2 against
Candida albicans ATCC 90028
a
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
measured with a Thomas-Hoover capillary melting point
apparatus and were uncorrected. UV spectra were measured
on a Hewlett-Packard 8453 spectrometer. IR spectra were
recorded on an ATI Mattson Genesis Series FTIR spectrom-
eter. NMR spectra were recorded with TMS as an internal
standard, using a Bruker Avance DRX-400 NMR spectrometer
for the H, C, and 2D NMR spectra. ESI-FTMS were
measured on a Bruker-Magnex BioAPEX 30es ion cyclotron
high-resolution HPLC-FT spectrometer by direct injection into
an electrospray interface. GC-MS was run on a Hewlett-
Packard (HP) 5890 Series II Plus gas chromatograph inter-
faced to a HP 5970B mass selective detector equipped with
Wiley Library for search: DB-1 Minibore capillary column
IC50(µg/mL)
MIC (µg/mL)
1
2
0.38 ( 0.03
0.33 ( 0.14
0.23 ( 0.06
0.18 ( 0.03
0.47 ( 0.12
1.04 ( 0.45
0.52 ( 0.23
0.52 ( 0.18
0.29 ( 0.00
1.04 ( 0.45
amphotericin B
fluconazole
flucytosine
1
13
a
Determined in triplicate.
tosine, with minimum inhibitory concentrations (MICs)
ranging from 0.29 to 1.04 µg/mL (Table 1). The correspond-
ing methylated products, 1a and 2a , were inactive against
C. albicans. Compounds 1 and 2 were not fungicidal to C.
albicans and did not show cytotoxicity in assays against
human tumor cells (KB, SK-MEL, BT-549, and SK-OV-3)
and Vero cells at concentrations of 10 µg/mL.
Compounds 1 and 2 were then evaluated for antifungal
activity against a series of C. albicans strains isolated from
(0.18 mm i.d., 20 m), column temperature 170f280 °C (10 °C/
minf25 min), He (45 psi). Column chromatography was run
using silica gel (40 µm, J . T. Baker) and reversed-phase silica
gel (RP-18, 40 µm, J . T. Baker). TLC was performed on silica
gel sheets (Alugram Sil G/UV254, Macherey-Nagel, Germany)
and reversed-phase plates (RP-18 F254S, Merck, Germany).
General procedures for antimicrobial and cytotoxicity assays
1
0
patients during fluconazole therapy with varying degrees
of fluconazole resistance (Table 2).2
,11-13
Isolate 17 has been
shown to possess several mechanisms of resistance to
fluconazole including overexpression (increased mRNA
levels) of CDR and MDR efflux pumps in addition to
overexpression of the target enzyme of fluconazole, lanos-
terol 14R-demethylase.12 Since compounds 1 and 2 were
equipotent in the fluconazole-susceptible (isolate 1) and
10,19,20
have been described in previous papers.
P la n t Ma ter ia l. The roots of P. gigantifolia Ducke were
collected by Mr. Manuel Rimachi in Loreto, Peru, in November
1996 and identified by Mr. M. Rimachi and Dr. Sidney
McDaniel. A voucher specimen is deposited at the Herbarium
of Mississippi State University (MISSA, voucher #IBE-MR
1
1821).
-
resistant (isolate 17) strains, it appears that 1 and 2 are
E xt r a ct ion a n d Isola t ion . Dried roots (500 g) of P.
not substrates for either the CDR or MDR efflux pumps.
In addition, it can be reasoned that the antifungal target
of 1 and 2 may not be lanosterol 14R-demethylase. Taking
into account the antifungal potency against resistant C.
albicans strains along with their selectivity and low
cytotoxicity, compounds 1 and 2 may be potential antifun-
gal leads for further studies.
Sphingolipid synthesis has been demonstrated to be an
antifungal target of sphingofungins,
and khafrefungin,18 which are structurally similar to
gigantifolia were percolated with 95% EtOH (3.5 L × 2).
Removal of the solvent under vacuum at 40 °C yielded an
EtOH extract (21.5 g; IC50 < 20 µg/mL against C. albicans). A
portion (5.3 g) of the EtOH extract was chromatographed on
silica gel (220 g) using a gradient solvent system consisting of
CHCl
several fractions. The fraction (0.93 g) that was eluted with
2% MeOH/CHCl and showed strongest activity (IC50 < 2 µg/
3
/MeOH (0% to 100% MeOH, a total of 8 L) to yield
3
1
4-16
17
mL) was further chromatographed on reversed-phase silica gel
(C18, 84 g) using 85%-90% MeOH (2 L) to give 6-octadecynoic
acid (1, 480 mg) and 6-nonadecynoic acid (2, 108 mg).
viridiofungins,
intermediates of the sphingolipid biosynthesis pathway.
Since compounds 1 and 2 are, to some extent, structurally
2
1
6
-Octa d ecyn oic a cid (1): white powder; mp 49 °C (lit.
mp 50-51 °C); UV (MeOH) λmax (log ꢀ) 204 (2.73) nm; IR (KBr)
similar to these known sphingolipid synthesis inhibitors,
ν
max 3102, 2916, 2849, 1707, 1692, 1469, 1316, 1264, 1207, 900,
a sphingolipid reversal assay1
4,17
was performed to explore
-1
1
7
3
18 cm ; H NMR (CDCl , 400 MHz) δ 0.88 (3H, t, J ) 7.0
their antifungal mechanism of action. The antifungal
activity of the two compounds was reversed with addition
of the sphingolipid synthesis intermediate, dihydrosphin-
gosine (concentrations at 0, 1.25, 2.5, and 5 µg/mL), to the
cell culture. This effect was not observed with the antifun-
gal drugs amphotericin B, fluconazole, or flucytosine (Table
Hz, H-18, HMBC/C-16,17), 1.26-1.37 (16H, m, H-10-17), 1.47
(2H, br quint, J ) 7.5 Hz, H-9, HMBC/C-7), 1.54 (2H, br quint,
J ) 7.2 Hz, H-4, HMBC/C-2,3,5,6), 1.74 (2H, br quint, J ) 7.6
Hz, H-3, HMBC/C-1,2,4,5), 2.13 (2H, tt, J ) 7.1, 2.2 Hz, H-8,
HMBC/C-5), 2.18 (2H, tt, J ) 7.0, 2.2 Hz, H-5, HMBC/C-
1
3
3
,4,6,7,8), 2.38 (2H, t, J ) 7.5 Hz, H-2, HMBC/C-1,3,4);
C
NMR (CDCl , 100 MHz) δ 14.5 (C-18), 18.8 (C-5), 19.1 (C-8),
3
3
), which all do not act on sphingolipid synthesis. However,
2
3
3.1 (C-17), 24.2 (C-3), 28.8 (C-4), 29.3, 29.5, 29.6, 29.8, 30.0,
0.02, 30.05, 32.3 (C-16), 34.0 (C-2), 79.7 (C-6), 81.3 (C-7), 180.6
stearylamine, which is structurally similar to dihydro-
sphingosine but not an intermediate of sphingolipid bio-
synthesis, also reversed the antifungal activity of com-
pounds 1 and 2 (Table 4). These preliminary results may
be helpful in future studies that include the use of these
compounds in microarray gene expression experiments to
examine their antifungal mechanism of action.
+
(
2
32 2
C-1); ESIMS m/z 281.2459 {calcd for [M(C18H O ) + H] ,
81.2475}.
-Non a d ecyn oic a cid (2): white powder; mp 48 °C; UV
MeOH) λmax (log ꢀ) 204 (2.70) nm; IR (KBr) νmax 3114, 2918,
6
(
-
1
1
2849, 1705, 1692, 1470, 1317, 1264, 1207, 900, 718 cm ; H
NMR (CDCl , 400 MHz) δ 0.87 (3H, t, J ) 7.0 Hz, H-19,
3
Ta ble 2. Antifungal Activity of Compounds 1 and 2 against Azole-Susceptible and -Resistant Candida albicans Strains [IC50/IC80/
a
IC95 (µg/mL)]
1
2
fluconazole
2
2
2
C. albicans isolate 1
C. albicans isolate 2
C. albicans isolate 5
0.45/0.60/0.70
0.50/0.65/0.75
0.70/1.00/2.00
0.90/1.00/1.50
0.60/0.90/1.00
0.65/1.00/1.50
0.25/0.35/0.45
0.25/0.30/0.35
0.45/0.60/0.80
0.50/0.65/0.80
0.40/0.50/0.70
0.45/0.60/0.70
1.00/1.50/2.00
1.00/5.00/10.00
7.50/10.00/10.00
15.00/20.00/25.00
0.10/0.20/100.00
2
C. alibicans isolate 8
1
1-13
C. albicans isolate 1
1
1-13
b
C. albicans isolate 17
40.00/95.00/NA
a
Patient isolates during fluconazole therapy: isolate 1, azole-susceptible strain; isolates 2, 5, 8, and 17, azole-resistant strains with
increasing azole resistance. b Not active at highest test concentration of 100 µg/mL.

1
134 J ournal of Natural Products, 2003, Vol. 66, No. 8
Notes
T. C. White.2,12,13 Antifungal susceptibility tests were per-
formed using a modified version of the NCCLS methods.
Ta ble 3. Antifungal Activity of Compounds 1 and 2 against
Candida albicans ATCC 90028 with Dihydrosphingosine (IC50,
µg/mL)
9
Compounds were dissolved in DMSO, serially diluted with
sterile normal saline, and transferred in duplicate to a flat-
bottomed 96-well microplate. Prior to the assay, C. albicans
growth from a Sabouraud dextrose agar (Difco, Detroit, MI)
slant was subcultured in 5 mL of Sabouraud dextrose broth
(Difco) at 37 °C for 24 h. The fungal inoculum was prepared
dihydrosphingosine (µg/mL)
0
1.25
2.5
5.0
1
2
0.40
0.25
0.30
0.15
0.40
0.70
1.0
0.10
0.30
0.20
2.0
2.0
0.10
0.50
0.15
4.5
4.5
0.10
0.70
0.10
9
amphotericin B
fluconazole
flucytosine
by comparison to the 0.5 McFarland standard and diluted in
RPMI 1640 without phenol red (2% glucose buffered with
MOPS at pH 7.0, Cellgro, Herndon, VA). The fungal inocula
were added to the samples, affording a final volume of 200
µL, final initial test concentration of 50 µg/mL, and final
inoculum size of approximately 2500 CFU/mL. Growth (saline
only), solvent (DMSO only), drug [amphotericin B (ICN Bio-
medicals, Aurora, OH), fluconazole (Pfizer, Morris Plains, NJ ),
and flucytosine], and blank (saline + broth) controls were
included in each determination. The plates were read at 630
nm using the EL-340 Biokinetics Reader (Bio-Tek Instru-
ments, Winooski, VT) prior to and after incubation (48 h at
Ta ble 4. Antifungal Activity of Compounds 1 and 2 against
Candida albicans ATCC 90028 with Stearylamine (IC50, µg/mL)
stearylamine (µg/mL)
0
5
15
15
15
0.40
0.80
0.20
30
30
35
0.65
0.75
0.25
1
2
0.40
0.25
0.30
0.15
0.40
3.5
5
amphotericin B
fluconazole
flucytosine
0.25
0.65
0.25
3
7 °C). Percent growth versus test concentration was calcu-
lated and plotted to afford the IC50, IC80, or IC95. The minimum
inhibitory concentration (MIC) was defined as the lowest test
concentration that allows no detectable growth (100% inhibi-
tion). Minimum fungicidal concentrations (MFCs) were deter-
mined by removing 5 µL of each duplicate at active test
concentration, transferring to Sabouraud dextrose agar, and
incubating at 37 °C for 24-48 h. The MFC is defined as the
lowest concentration of sample that allows no growth on agar.
HMBC/C-17,18), 1.25-1.37 (18H, m, H-10-18), 1.46 (2H, br
quint, J ) 7.3 Hz, H-9, HMBC/C-7), 1.54 (2H, br quint, J )
7
.3 Hz, H-4, HMBC/C-2,3,5,6), 1.74 (2H, br quint, J ) 7.6 Hz,
H-3, HMBC/C-1,2,4,5), 2.12 (2H, tt, J ) 7.1, 2.2 Hz, H-8,
HMBC/C-5), 2.17 (2H, tt, J ) 7.0, 2.2 Hz, H-5, HMBC/C-
3
,4,6,7,8), 2.38 (2H, t, J ) 7.5 Hz, H-2, HMBC/C-1,3,4); 13
C
Sp h in golip id Rever sa l Assa y. The sphingolipid reversal
NMR (CDCl
3
, 100 MHz) δ 14.5 (C-19), 18.8 (C-5), 19.1 (C-8),
14,17
assay
was performed as above for C. albicans ATCC 90028.
2
3
1
3.1 (C-18), 24.2 (C-3), 28.8 (C-4), 29.3, 29.5, 29.6, 29.8, 30.0,
However, the RPMI broth was supplemented with varying
concentrations of DL-dihydrosphingosine or stearylamine (both
from Sigma, St. Louis, MO), initially dissolved in a solution
of ethanol and methyl â-cyclodextrin. Maximum final test
concentrations of ethanol and â-cyclodextrin were 1.56% and
0.1 (×2), 30.2, 32.3 (C-16), 34.0 (C-2), 79.7 (C-6), 81.3 (C-7),
80.3 (C-1); ESIMS m/z 295.2622 {calcd for [M(C19
34 2
H O ) +
+
H] , 295.2632}.
Meth yla tion of 1 a n d 2. A solution of each compound (20
mg) in BF /MeOH (14% w/v, 0.6 mL) was heated at 100 °C for
min. After cooling, the reaction mixture was diluted with
O (1 mL) and then extracted with hexanes (10 mL × 3).
O (5 mL),
. Removal of the solvent yielded me-
3
1
25 µg/mL, respectively, and were not inhibitory to the growth
2
H
of the fungus.
2
The hexane layers were combined, washed with H
and dried over Na SO
2
Ack n ow led gm en t. The authors wish to thank Dr. Ted
White and Dr. Spencer Redding for kindly providing the
fluconazole-resistant C. albicans strains and to Dr. D. Chuck
Dunbar for running ESIMS. We thank Mr. J ohn M. Trott, Ms.
Marinda Logan, Ms. Sharon Sanders, and Ms. Belynda Smiley
for biological testing and technical assistance. This work was
supported in part by a grant from the National Institutes of
Health, National Institute of Allergy and Infectious Diseases,
Division of AIDS, Grant No. AI 27094, and by the United
States Department of Agriculture, Agricultural Research
Service Specific Cooperative Agreement No. 58-6408-2-0009.
2
4
thylated products 1a and 2a (ca. 20 mg each), respectively.
1
Meth yl 6-octa d ecyn oxyla te (1a ): colorless oil; H NMR
(
(
(
3
CDCl , 400 MHz) δ 0.88 (3H, t, J ) 7.0 Hz, H-18), 1.26-1.37
16H, m, H-10-17), 1.46 (2H, br quint, J ) 7.0 Hz, H-9), 1.51
2H, br quint, J ) 7.0 Hz, H-4), 1.72 (2H, br quint, J ) 7.5
Hz, H-3), 2.12 (2H, tt, J ) 7.1, 2.3 Hz, H-8), 2.17 (2H, tt, J )
7
COOMe); C NMR (CDCl
5
2
.1, 2.3 Hz, H-5), 2.33 (2H, t, J ) 7.5 Hz, H-2), 3.67 (3H,
13
3
, 100 MHz) δ 14.5 (C-18), 18.8 (C-
), 19.1 (C-8), 23.1 (C-17), 24.5 (C-3), 28.9 (C-4), 29.3, 29.5,
9.6, 29.7, 29.9, 30.0 (×2), 32.3 (C-16), 34.0 (C-2), 51.8
) 13.6
dC(OH)-
COOMe] , 122 (26) [154
(
COOMe), 79.7 (C-6), 81.1 (C-7), 174.4 (C-1); GC-MS (t
R
+
min) m/z 263 (1.8) [M - OMe] , 220 (3.9) [M - CH
OMe] , 154 (53) [CH
2
Refer en ces a n d Notes
+
+
2
2 4
dCdCH-(CH )
(
1) Pfaller, M. A.; J ones, R. N.; Messer, S. A.; Edmond, M. B.; Wenzel,
+
+
-
HOMe] , 94 (95) [122 - CO] , 80 (100) [154 - CH
2
dC(OH)-
R. P. Diagn. Microbiol. Infect. Dis. 1998, 31, 327-332.
+
+
OMe] , 67 (48) [94 - (CH
2
)
2
+ H] .
(2) Marr, K. A.; Lyons, C. N.; Rustad, T. R.; Bowden, R. A.; White, T. C.
1
Antimicrob. Agents Chemother. 1998, 42, 2584-2589.
Meth yl 6-n on a d ecyn oxyla te (2a ): colorless oil; H NMR
(
(
(
3) White, T. C.; Marr, K. A.; Bowden, R. A. Clin. Microbiol. Rev. 1998,
(
(
(
3
CDCl , 400 MHz) δ 0.86 (3H, t, J ) 7.0 Hz, H-19), 1.24-1.37
1
1, 382-402.
18H, m, H-10-18), 1.45 (2H, br quint, J ) 7.0 Hz, H-9), 1.50
2H, br quint, J ) 7.0 Hz, H-4), 1.71 (2H, br quint, J ) 7.6
4) White, T. C.; Holleman, S.; Dy, F.; Mirels, L. F.; Stevens, D. A.
Antimicrob. Agents Chemother. 2002, 46, 1704-1713.
5) Marr, K. A.; Lyons, C. N.; Ha, K.; Rustad, T. R.; White, T. C.
Antimicrob. Agents Chemother. 2001, 45, 52-59.
Hz, H-3), 2.11 (2H, tt, J ) 7.1, 2.2 Hz, H-8), 2.16 (2H, tt, J )
7
COOMe); C NMR (CDCl
5
2
7
2
.1, 2.2 Hz, H-5), 2.32 (2H, t, J ) 7.4 Hz, H-2), 3.65 (3H,
(
(
6) Purcell, J . M.; Susi, H. Anal. Chem. 1968, 40, 571-575.
7) Pearl, M. B.; Kleiman, R.; Earle, F. R. Lipids 1974, 8, 627-630.
13
3
, 100 MHz) δ 14.5 (C-18), 18.9 (C-
), 19.1 (C-8), 23.1 (C-17), 24.5 (C-3), 29.0 (C-4), 29.3, 29.6,
9.8, 30.0, 30.1 (×4), 32.3 (C-16), 34.0 (C-2), 51.9 (COOMe),
9.8 (C-6), 81.1 (C-7), 174.4 (C-1); GC-MS (t ) 16.5 min) m/z
(8) Valicenti, A. J .; Heimermann, W. H.; Holman, R. T. J . Org. Chem.
1
979, 44, 1068-1073.
(
9) National Committee for Clinical Laboratory Standards. Reference
Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts.
Approved Standard M27-A, Vol. 17 (9). National Committee for
Clinical Laboratory Standards: Wayne, PA, 1997; 4th ed.
R
+
+
2
77 (1.6) [M - OMe] , 234 (4.3) [M - CH dC(OH)OMe] , 154
+
+
(62) [CH
2
2 4
dCdCH-(CH ) COOMe] , 122 (26) [154 - HOMe] ,
+
+
(10) Muhammad, I.; Dunbar, D. C.; Takamatsu, S.; Walker, L. A.; Clark,
9
(
2
4 (95) [122 - CO] , 80 (100) [154 -CH dC(OH)OMe] , 67
A. M. J . Nat. Prod. 2001, 64, 559-562.
+
44) [94 - (CH
2 2
) + H] .
(
11) Pfaller, M. A.; Rhine-Chalberg, J .; Redding, S. W.; Smith, J .; Farinacci,
G.; Fothergill, A. W.; Rinaldi, M. G. J . Clin. Microbiol. 1994, 32, 59-
64.
An tifu n ga l Su scep tibility Testin g. All chemicals were
obtained from Sigma Chemical Company (St. Louis, MO)
unless otherwise stated. C. albicans ATCC 90028 was pur-
chased from ATCC (Rockville, MD). Azole-resistant C. albicans
(
12) White T. C. Antimicrob. Agents Chemother. 1997, 41, 1482-
1
487.
(
13) White T. C. Antimicrob. Agents Chemother. 1997, 41, 1488-
1
1
strains were kindly provided by Dr. S. W. Redding and Dr.
1494.

Notes
J ournal of Natural Products, 2003, Vol. 66, No. 8 1135
(
14) Zweerink, M. M.; Edison, A. M.; Wells, G. B.; Pinto, W.; Lester, R. L.
(18) Mandala, A. M.; Thornton, R. A.; Rosenbach, M.; Milligan, J .; Garcia-
Calvo, M.; Bull, H. G.; Kurtz, M. B. J . Biol. Chem. 1997, 272, 32709-
32714.
(19) Li, X.-C.; ElSohly, H. N.; Nimrod, A. C.; Clark, A. M. J . Nat. Prod.
1999, 62, 767-769.
(20) Li, X.-C.; Dunbar, C.; ElSohly, H. N.; J acob, M. R.; Nimrod, A. C.;
Walker, L. A.; Clark, A. M. J . Nat. Prod. 2001, 64, 1153-1156.
(21) Lumb, P. B.; Smith, J . C. J . Chem. Soc. 1952, 5032-5035.
J . Biol. Chem. 1992, 267, 25032-25038.
(
15) Horn, W. S.; Smith, J . L.; Bills, G. F.; Raghoobar, S. L.; Helms, G.
L.; Kurtz, M. B.; Marrinan, J . A.; Frommer, B. R.; Thornton, R. A.;
Mandala, S. M. J . Antibiot. 1992, 45, 1692-1696.
(
16) Mandala, A. M.; Frommer, B. R.; Thornton, R. A.; Kurtz, M. B.; Young,
N. M.; Gabello, M. A.; Genilloud, O.; Liesch, J . M.; Smith, J . L.; Horn,
W. S. J . Antibiot. 1994, 47, 376-379.
(17) Mandala, A. M.; Thornton, R. A.; Frommer, B. R.; Dreikorn, S.; Kurtz,
M. B. J . Antibiot. 1997, 50, 339-343.
NP030196R